Protein-Ligand Interaction Analyses with Nuclear Magnetic Resonance Spectroscopy Enhanced by Dissolution Triplet Dynamic Nuclear Polarization.

Solution-state nuclear magnetic resonance spectroscopy (NMR) is a powerful method for the analysis of intermolecular interactions within a biomolecular system. However, low sensitivity is one of the major obstacles of NMR. We improved the sensitivity of solution-state 13C NMR for the observation of intermolecular interactions between protein and ligand using hyperpolarized solution samples at room temperature. Eutectic crystals composed of 13C-salicylic acid and benzoic acid doped with pentacene were hyperpolarized by dynamic nuclear polarization using photoexcited triplet electrons, and a 13C nuclear polarization of 0.72 ± 0.07% was achieved after dissolution. The binding of human serum albumin and 13C-salicylate was observed with several hundred times sensitivity enhancement under mild conditions. The established 13C NMR was applied for pharmaceutical NMR experiments by observation of the partial return of the 13C chemical shift of salicylate by competitive binding with other non-isotope-labeled drugs.

Safety of tapering tacrolimus dose in patients with well-controlled anti-acetylcholine receptor antibody-positive myasthenia gravis.

BACKGROUND AND PURPOSE: Tapering immunosuppressants is desirable in patients with well-controlled myasthenia gravis (MG). However, the association between tapering of calcineurin inhibitor dosage and reduction-associated exacerbation is not known. The aim of this study was to clarify the frequency of reduction-associated exacerbation when tacrolimus is tapered in stable patients with anti-acetylcholine receptor antibody-positive MG, and to determine the factors that predict exacerbations. METHODS: We retrospectively analyzed 115 patients in whom tacrolimus dosage was tapered. The reduction-associated exacerbation was defined as the appearance or worsening of one or more MG symptoms <3 months after the reduction. RESULTS: Tacrolimus dosage was successfully tapered in 110 patients (96%) without any exacerbation. Five patients (4%) experienced an exacerbation, but symptoms were reversed in all patients when the tacrolimus dose was increased to the previous maintenance level. No patient developed an MG crisis. The age at onset was significantly earlier (30 vs. 56 years, P = 0.025) and the reduction in dosage was significantly larger (2.0 vs. 1.0 mg/day, P = 0.002) in patients with reduction-associated exacerbation than in those without exacerbation. The cut-off values determined in a receiver-operating characteristic curve analysis were 52 years (sensitivity, 57%; specificity, 100%) for the age at onset and 1.5 mg (sensitivity, 80%; specificity, 100%) for the dose reduction. CONCLUSION: Tapering of tacrolimus was possible in most patients with well-controlled anti-acetylcholine receptor antibody-positive MG. Early age at onset and a large reduction from maintenance dosage were associated with exacerbation. Reductions ≤1.5 mg/day from the maintenance dosage should be considered for patients with late-onset disease.

Prognostic significance of epithelial-mesenchymal transition-related markers in extrahepatic cholangiocarcinoma: comprehensive immunohistochemical study using a tissue microarray.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is characterised by the loss of cell-to-cell adhesion and gaining of mesenchymal phenotypes. Epithelial-mesenchymal transition is proposed to occur in various developmental processes and cancer progression. 'Cadherin switch', a process in which cells shift to express different isoforms of the cadherin transmembrane protein and usually refers to a switch from the expression of E-cadherin to N-cadherin, is one aspect of EMT and can have a profound effect on tumour invasion/metastasis. The aim of this study was to investigate the clinicopathological significance of EMT-related proteins and cadherin switch in extrahepatic cholangiocarcinoma (EHCC). METHODS: We investigated the association between altered expression of 12 EMT-related proteins and clinical outcomes in patients with EHCC (n=117) using immunohistochemistry on tissue microarrays. RESULTS: Univariate and multivariate analyses revealed that, in addition to N classification (P=0.0420), the expression of E-cadherin (P=0.0208), N-cadherin (P=0.0038) and S100A4 (P=0.0157) was each an independent and a significant prognostic factor. We also demonstrated that cadherin switch was independently associated with poor prognosis (P=0.0143) in patients with EHCC. CONCLUSIONS: These results may provide novel information for selection of patients with EHCC who require adjuvant therapy and strict surveillance.

Sudden onset of sleep due to hypothalamic lesions in neuromyelitis optica spectrum disorder positive for anti-aquaporin-4 antibody.

We report a patient with neuromyelitis optica spectrum disorders who presented with sudden onset of sleep as the sole manifestation. Magnetic resonance imaging investigation revealed lesions in the hypothalamus bilaterally, which vanished completely after methylprednisolone pulse therapy.

Effects of additional cysteine in fish diet on mercury concentration.

Mercury contamination, especially of seafood, continues to attract public concern. Cysteine, NH2CH(CH2SH)COOH, is a naturally occurring hydrophobic amino acid that contains a thiol group. The purpose of our study was to investigate the use of the additive cysteine in fish diets to reduce mercury concentration in fish, and to observe the effectiveness of dietary cysteine in fish livers. Diets containing 1% and 10% cysteine successfully decreased mercury concentrations in fish compared with the 0% cysteine diet. The liver may have formed excessive lipid droplets or was unable to mobilize lipid stores during exposure to mercury; additional cysteine could help to mobilize excessive lipids in it.

IFNß-1b may severely exacerbate Japanese optic-spinal MS in neuromyelitis optica spectrum.

BACKGROUND: Interferon-ß-1b (IFNß-1b) has been used to prevent exacerbation of relapsing-remitting multiple sclerosis (RRMS) including optic-spinal multiple sclerosis (OSMS) in Japan. We encountered 2 patients with OSMS with unexpectedly severe exacerbation soon after the initiation of IFNß-1b therapy. The experience urged us to retrospectively review the patients with RRMS who had been treated with IFNß-1b to identify similar cases. METHODS: At neurologic departments of 9 hospitals, the medical records of 56 patients with RRMS were reviewed to identify those who showed severe exacerbation soon after the initiation of IFNß-1b therapy. RESULTS: Of 56 patients with RRMS, we identified 7 who experienced severe exacerbation (exacerbation with increased scores of Expanded Disability Status Scale ≧7.0) within 90 days of the initiation of IFNß-1b therapy. In all 7 patients, the exacerbations after the initiation of IFNß-1b therapy were more severe than those experienced by the individual patients before the use of IFNß-1b, and seemed to have occurred unexpectedly in a short time after the initiation of INFß-1b therapy. A retrospective analysis revealed that all 7 patients had antibodies toward aquaporin 4, and the clinical features of all 7 patients after the exacerbation were consistent with those of neuromyelitis optica (NMO) spectrum. CONCLUSIONS: Our study suggests that IFNß-1b may trigger severe exacerbation in patients with the NMO spectrum. In INFß-1b therapy, cases in NMO spectrum should be carefully excluded.

Localization of latexin-immunoreactive neurons in the adult cat cerebral cortex and claustrum/endopiriform formation.

The distribution of neurons that are immunoreactive to latexin, which is an endogenous inhibitor of the A/B subfamily of metallocarboxypeptidases, was investigated in the adult cat telencephalon. Latexin-immunoreactive neurons were distributed in the lower layers of the neocortex and adjacent ventral mesocortex, as well as in the claustrum/endopiriform formation. There were marked regional and laminar differences in density and distribution of latexin-immunoreactive neurons in the cerebral cortex. The density followed a roughly lateral-to-medial decreasing gradient: it was high in lateral cortical regions, which included the insular, second somatosensory, and anterior sylvian areas, and in the temporal auditory field; moderate in laterodorsal cortical regions, which included the primary and second auditory fields; and low in dorsal cortical regions, which included visual areas 18 and 19. Latexin-immunoreactive neurons were absent in medial cortical regions, which included the motor, premotor, prefrontal, prelimbic, cingulate, and retrosplenial areas. The lateral-to-medial gradient was apparent even within a single cytoarchitectonic area in certain cortical regions. The allocortex was devoid of latexin-immunoreactive neurons, with the exception of the anteroventral part of the dentate gyrus. The majority of cortical latexin-immunoreactive neurons were localized in layers V and VI and appeared to correspond to the "modified pyramidal cells in the infragranular layers." The remaining latexin-immunoreactive neurons were localized in layer IV, as well as in lower layer III and in the white matter. There were no latexin-immunoreactive neurons from layer I through upper layer III. Latexin-immunoreactive neurons were present in telencephalic structures outside the cerebral cortex, with particularly high density in the claustrum/endopiriform formation. All these features, with the exception of that detected in the archicortex, are compatible with the features observed previously in the rat telencephalon. The similar pattern of distribution of latexin-immunoreactive neurons in several mammalian species from different orders suggests that latexin plays an important role in a specific cortical network.

A ketolide antibiotic, telithromycin, inhibits vascular adrenergic neurotransmission in the rat mesenteric vascular bed.

BACKGROUND AND PURPOSE: A ketolide antibiotic, telithromycin, has side effects including temporary loss of consciousness in clinical use, but the underlying mechanisms remain unclear. This study investigated the effects of telithromycin on perivascular nerve function in rat mesenteric arteries, in comparison with those of macrolide (erythromycin and clarithromycin) and new quinolone antibiotics (levofloxacin and gatifloxacin). EXPERIMENTAL APPROACH: In vitro, vascular responses and release of noradrenaline induced by periarterial nerve stimulation (PNS) of rat perfused mesenteric vascular beds were measured in the presence of each antibiotic. In vivo blood pressure measurement was performed in Wistar rats. KEY RESULTS: In mesenteric preparations with resting tone, telithromycin (10 nM-10 microM) markedly inhibited PNS (4-12 Hz)-induced adrenergic nerve- and exogenous noradrenaline-mediated vasoconstriction, whereas the other antibiotics slightly inhibited PNS-induced responses without affecting noradrenaline-induced responses. Telithromycin significantly reduced PNS (12 Hz)-evoked noradrenaline release in the perfusate. In pre-constricted preparations with or without endothelium, telithromycin (0.1 nM-10 microM) caused a concentration-dependent vasodilation. Telithromycin (10 nM) inhibited calcium-induced vasoconstriction in high KCl and calcium-free medium. None of the antibiotics used affected PNS (0.5-2 Hz)-induced calcitonin gene-related peptide (CGRP) nerve- and exogenous CGRP-mediated vasodilation. Intravenous injection of telithromycin significantly lowered blood pressure in anaesthetized rats. CONCLUSIONS AND IMPLICATIONS: These results suggest that telithromycin causes not only strong inhibition of perivascular adrenergic neurotransmission but also a vasodilator action in mesenteric vascular beds and hypotension. It is thus possible that telithromycin increases visceral blood flow, consequently reducing cerebral blood flow and resulting in a temporary loss of consciousness.

Cotransduction of CCL27 gene can improve the efficacy and safety of IL-12 gene therapy for cancer.

Interleukin-12 (IL-12) is a potent antitumoral cytokine, but high doses are toxic. Herein, we demonstrate that combinational transduction of IL-12 and CC-chemokine ligand-27 (CCL27) genes into pre-existing murine OV-HM ovarian carcinoma and Meth-A fibrosarcoma, by using RGD fiber-mutant adenoviral vectors, could induce tumor regression and relieve systemic side effects more effectively than either treatment alone. The antitumor activity of the IL-12 and CCL27 combination treatment was T-cell-dependent, and development of long-term specific immunity was confirmed in rechallenge experiments. Immunohistochemical analysis of tumors transduced with CCL27 gene alone or cotransduced with IL-12 and CCL27 genes showed significant increases in numbers of infiltrating CD3(+) T cells, which included both CD4(+) and CD8(+) cells. Additionally, cotransduction with IL-12 and CCL27 genes could more efficiently activate tumor-infiltrating immune cells than transduction with CCL27 alone, as determined by the frequency of perforin-positive cells and expression levels of IFN-gamma. Furthermore, mice treated with the IL-12 and CCL27 combination compared with those treated with IL-12 alone showed milder pathological changes, for example, lymphocyte infiltration and extramedullary hematopoiesis, in lung, liver and spleen. Our data provide evidence that combinational in vivo transduction with IL-12 and CCL27 genes is a promising approach for the development of cancer immunogene therapy that can simultaneously recruit and activate tumor-infiltrating immune cells.

Spread of the radial SNAP: a pitfall in the diagnosis of carpal tunnel syndrome using standard orthodromic sensory conduction study.

OBJECTIVE: To investigate the occurrence of the spread of the radial sensory nerve action potential (SNAP) among patients with carpal tunnel syndrome (CTS) during standard median orthodromic sensory conduction study (SCS) using index finger stimulation. METHODS: We prospectively examined 74 hands in 56 CTS patients. We stimulated the index finger using ring electrodes. SNAPs were recorded at wrist over median and radial nerves. RESULTS: A spread of radial SNAP was clearly identified over the median nerve despite its small amplitude, in 72/74 hands during stimulation of the base of the index finger. In hands with delayed median SNAP, two peaks were observed; however in hands with absence of genuine median SNAP, only one peak of the spread was noticed. The proximal interphalangeal joint (PIP) stimulation still elicited an identifiable spread in 47/74 hands. CONCLUSION: This spread phenomenon is a previously undescribed pitfall during the standard median orthodromic SCS, frequently occurring in CTS patients. SIGNIFICANCE: In severe CTS cases, one may make wrong conclusion of normal median sensory latency if unaware of this pitfall.

Tumor suppressive efficacy through augmentation of tumor-infiltrating immune cells by intratumoral injection of chemokine-expressing adenoviral vector.

Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cell-based immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.

Vasodilative effect of perillaldehyde on isolated rat aorta.

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.

N10 component in median nerve somatosensory evoked potentials (SEPs) is not an antidromic motor potential.

OBJECTIVE: To test the hypothesis that the N10 far-field potential in median nerve somatosensory evoked potentials is generated by the motor axons by examining patients with amyotrophic lateral sclerosis (ALS). METHODS: Subjects were 5 ALS patients showing pronounced or complete denervation of median-innervated small hand muscles. We evaluated N10 over scalp, and proximal plexus volleys (PPVs) at lateral or anterior cervical electrode. RESULTS: N10 and PPVs were definitely preserved for every ALS subject. N10 amplitudes of ALS subjects were even significantly larger than control subjects. In one ALS patient completely lacking motor axons, N10 was larger than the largest one among control subjects. CONCLUSIONS: Present results clearly indicate that N10 is not predominantly generated by motor axons but by the whole median nerve dominated by sensory axons. We propose a theory that N10 is a junctional potential generated by the entrance of the median nerve into bone at the intervertebral foramen, producing a positive pole at the non-cephalic reference electrode. Significantly larger N10 in ALS subjects may be due to the lack of cancellation by slower motor axons. SIGNIFICANCE: The hypothesis that N10 is generated by motor axons is refuted, and a new theory of its generation is presented.

Postrenal acute renal failure during pregnancy 20 years after antireflux surgery.

We report that a 27-year-old woman with bilateral severe hydronephorosis during pregnancy 20 years after antireflux surgery. The patient developed postrenal acute renal failure due to obstruction of the lower ureter. This patient could safely give birth after bilateral percutaneous nephrostomy through joint management with the obstetrics and gynecology staff. We describe that stenosis of the lower ureter is a late complication of antireflux surgery.

Decreased expression of Bcl-x protein during hepatocarcinogenesis induced exogenously and endogenously in rats.

Dysregulations of apoptosis have been widely recognized as important events in multi-stage carcinogenesis. Bcl-x, a member of the Bcl-2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl-x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short-term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg / kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S-transferase (GST-P)-positive, putative preneoplastic lesions were induced, and Bcl-x protein expression was decreased in 24.7% of such lesions. The incidence of GST-P-positive lesions with decreased Bcl-x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm(2), respectively. In GST-P-positive lesions larger than 0.3 mm(2), both apoptosis induction and cell proliferation activity were enhanced when Bcl-x protein expression was decreased. In the long-term exogenous models, rats were given 10 mg / kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg / kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl-x protein expression. In the endogenous model, rats were fed a choline-deficient, L-amino acid-defined diet for 16 or 80 weeks and sacrificed. Bcl-x protein expression was decreased both in GST-P-positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl-x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.

Effects of antihypertensive drugs and gene variants in the renin-angiotensin system.

Many genes and environmental factors are involved in the pathogenesis of hypertension, but the exact cause of essential hypertension has not yet been clarified. Gene polymorphism of the renin-angiotensin system (RAS) is one of the candidates. In the current study, we examined whether there was a correlation between the gene polymorphisms in RAS and either the choice of antihypertensive drugs or their efficacy. Subjects with essential hypertension (n=299) were recruited from among the outpatients of Osaka University Hospital and provided their informed consent for genetic analysis. Physicians freely chose the antihypertensive drugs and adjusted its dose until the patient's blood pressure was well controlled. The efficacy of each antihypertensive drug was estimated using the following formula: ABP=BP 1 (before treatment) - BP 2 (after treatment)/BP 1 x 100 (%). Gene variants in RAS were determined using PCR or PCR-RFLP (restriction fragment of polymorphism). The gene polymorphisms of RAS were not associated with delta SBP or ADBP. However, the mean ASBP in subjects with a deletion homozygote of the angiotensin converting enzyme gene (ACE/DD) was significantly lower (p<0.05) than that in patients with an insertion I allele of the ACE gene. The gene polymorphisms of RAS did not significantly affect the choice of antihypertensive drugs. Even though gene polymorphism in the renin angiotensin system was not a major factor in the antihypertensive therapy, the determination of genotype might be of help in the management of essential hypertension.

Lower cervical origin of the P13-like potential in median SSEPS.

The authors studied the origin of the scalp P13-like potential in median somatosensory evoked potentials, which have been reported to be preserved in patients with cervicomedullary lesions or in brain death. There were five patients with high to middle cervical lesions (C2/3 or C3/4 level). Small P13-like potentials after P11 were identified for all patients with a noncephalic reference but not with an ear reference. Their onset latencies were slightly earlier than the expected latency of the true P13/14 onset. In two patients, delayed true P13/14s followed by N18s were identified with both noncephalic and ear references. The authors argue that the P13-like potential observed in these patients is a different entity from scalp P13 in normal subjects. Because the C3/4 vertebral level corresponds to the C5 cord level, the origin of the P13-like potential must be below C5, contradicting the previous opinion that it is generated at the cervicomedullary junction or at the high cervical dorsal column. The authors named this potential lower cervical P13 (or lcP13), and present an opinion that it is generated by the beginning of the second spinal ascending volley, which has been described by direct-recording studies in humans.

Angiotensin-converting enzyme inhibition with enalapril slows progressive intima-media thickening of the common carotid artery in patients with non-insulin-dependent diabetes mellitus.

BACKGROUND AND PURPOSE: Whether angiotensin-converting enzyme (ACE) inhibitors have any clinically significant antiatherogenic effects in humans remains unproven. We undertook a prospective randomized clinical trial of 98 patients with non-insulin-dependent diabetes mellitus (NIDDM) to examine the efficacy of ACE inhibition with enalapril for preventing intima-media (IM) thickening of the carotid wall as measured ultrasonographically. METHODS: Ninety-eight NIDDM patients were randomly assigned either to enalapril at 10 mg/d (n=48) or to a control group (n=50); the planned duration of the trial was 2 years. All patients were seen at baseline (study entry) and 2 subsequent formal annual evaluations, in addition to standard clinical management for NIDDM. IM thickening and vascular lumen diameters were determined for all patients on the basis of baseline and 2 subsequent annual evaluations with carotid ultrasonography. We performed an intent-to-treat analysis to assess changes in IM thickening over the course of the study. RESULTS: Annual IM thickening measurements of the right and left common carotid arteries were 0.01+/-0.02 and 0.01+/-0.02 mm/y in the enalapril-treated group and 0.02+/-0.03 and 0.02+/-0.02 mm/y in the control group, respectively (P<0.05). From regression analysis, annual IM thickening was found to be predicted by enalapril use, sex, and insulin use (F(3,94)=3.86, P=0.012). When we controlled for these other variables, enalapril use reduced annual IM thickening of right and left common carotid arteries by 0.01+/-0.004 mm/y relative to the control group over the course of this study. CONCLUSIONS: Long-term treatment with an ACE inhibitor (enalapril) slows progressive IM thickening of the common carotid artery in NIDDM patients.